The development of nucleic acid amplification methodologies, for instance the polymerase chain reaction (PCR), enables the use of DNA amplification for a variety of uses, including molecular diagnostic testing. There are challenges associated with the use of PCR for molecular differential diagnostic (MDD) assays, however. PCR utilizes specific primers or primer sets, temperature conditions, and enzymes. For example, PCR reactions are easily contaminated, primer binding may require different conditions for different primers and primers should be specific for a target sequence in order to amplify only that target sequence. These limitations make it even more difficult to amplify multiple sequences from a single sample.
Diagnostic testing of clinical samples to find one or more causative disease agents has, in the past, required that microorganisms be isolated and cultured. This may take days, however, and in many cases a diagnosis must be acted upon within hours if the patient's life is to be saved. Analysis of a single clinical sample to identify multiple organisms in order to determine which one(s) may be the causative agent(s) of disease is the desired method for MDD, and methods have been developed to better achieve that goal. For example, multiplex PCR methods have been developed to amplify multiple nucleic acids within a sample in order to produce enough DNA/RNA to enable detection and identification of multiple organisms. Multiplex PCR has disadvantages, however. For example, each target in a multiplex PCR reaction requires its own optimal reaction conditions, so increasing the number of targets requires that the reaction conditions for each individual target are less than optimal. Furthermore, multiple sets of high-concentration primers in a system often generate primer dimmers or give non-specific, background amplification. This lack of specificity also requires the additional steps of post-PCR clean-up and multiple post-hybridization washes.
Crowded primers reduce amplification efficiency by requiring the available enzymes and consuming substrates. Differences in amplification efficiency may lead to significant discrepancies in amplicon yields. For example, some loci may amplify very efficiently, while others amplify very inefficiently or fail to amplify at all. This potential for uneven amplification also makes it difficult to impossible to accurately perform end-point quantitative analysis.
Often, time is of the essence, patient infections include more than one bacterial species, and the amount of the target DNA in a clinical sample is limiting. Technologies such as multiplex PCR have been developed to address these issues. However, improvements in this field are still needed in order to further decrease the time and effort required to accomplish quick and accurate analysis of clinical samples. Methods that can be automated, especially by use of a closed cassette for sample preparation and analysis, are particularly important, as they may provide the added advantages of decreasing potential contamination of samples and decreasing the time and effort that a laboratory technician must invest in preparing and analyzing each sample.
Although there have been significant improvements in multiplex sequencing technologies and their use in sample analysis, it would be highly beneficial if these could be further refined to make analysis and detection easier.